Invasion and colonization of mature apple fruit by Erwinia amylovora tagged with bioluminescence genes

Invasion and colonization of mature apple fruit by a transformant of Erwinia amylovora tagged with bioluminescence genes from Vibrio fischeri was examined. The transformant was deposited on cut surfaces of fruit stems, wounds on the shoulders and calyces, injured fruit-bearing twigs of harvested apple fruit, and cut fruit flesh. After incubation in closed stainless steel or plastic boxes at 25°C, fruit were periodically observed with a two-dimensional luminometer. The presence of the transformant in luminous areas was confirmed by isolating it on selective media. E. amylovora, when deposited in fruit stems: (1) can invade mature as well as immature apple fruit; (2) vertically and horizontally spreads and colonizes along vascular bundles, increasing its population; (3) reaches the calyx end and the flesh just under the exocarp within 3–4 days after inoculation; (4) when deposited on cut fruit flesh, irrespective of its maturity, can easily increase its population and survive 2–4 weeks or more at 25°C; and (5) even at the time of fruit maturation, can migrate within twigs rapidly and reaches the abscission layers between fruit-bearing twigs and fruit stems.     

Malignant melanoma of the eyelid in a red squirrel (Sciurus vulgaris orientis)

A 6-year-old male red squirrel (Sciurus vulgaris orientis) developed bilateral tumors of upper and lower eyelids. The tumors in the left lid recurred despite surgical removal. Necropsy revealed metastasis to the lung. The neoplastic cells were epithelioid and highly pleomorphic, and only a few cells contained melanin granules. Occasionally melanoma cells were immunoreactive for S100, neuron-specific enolase and vimentin, and a small number of cells for cytokeratin. Ultrastructurally, the presence of premelanosomes was confirmed in the cytoplasm. Possible presence of cytokeratin-positive neoplastic melanocytes should be taken into account when differentiating a nonpigmented epithelioid melanoma from other tumors such as anaplastic carcinomas.     

Similarity between Copper Resistance Genes from Pseudomonas syringae pv. actinidiae and P. syringae pv. tomato

Twenty-eight strains of Pseudomonas syringae pv. actinidiae isolated in 1984, 1987 and 1988 from kiwifruit orchards in Japan were tested for their resistance to copper sulfate. All strains isolated in 1984 were copper sensitive with a minimum inhibitory concentration (MIC) of cupric sulfate of 0.75 mM. However, some strains isolated in 1987 and 1988 were resistant, with the MIC ranging from 2.25 to 3.0 mM. All copper-resistant strains contained at least one of two plasmids, pPaCul (about 70.5 kb) or pPaCu2 (about 280 kb), or both. In a copper-resistant strain Pa429, the location of the copper-resistance gene(s) was examined by insertional inactivation with Tn5. The MIC of copper sulfate in the copper-sensitive mutant obtained by Tn5 tagging decreased from 2.75 to 0.75 mM. The 14.5 kb BamHI fragment, designated pPaCuB14, containing the same locus mutagenized with Tn5 was cloned from pPaCu1. However, pPaCuB14 did not confer copper resistance in the transformant of copper-sensitive strain Pa21R, suggesting that this clone did not contain a full set of copper-resistance gene(s). Then a cosmid library of pPaCu1 was constructed and six cosmid clones hybridized with pPaCuB14 were selected. One of the six cosmids, designated pPaCuC1, conferred a near wild-type level of copper resistance in the transformant of the copper-sensitive strain. pPaCuC1 had a homologous region that hybridized with all of the PCR-amplifled fragments of copA, copB, copR, and copS genes of P. syringae pv. tomato. DNA sequence analysis of the homologous region revealed the existence of four open reading frames (ORF A, B, R and S) oriented in the same direction. The predicted amino acid sequences of ORF A, B, R and S had 80, 70, 97 and 95% identity with CopA, B, R and S of P. syringae pv. tomato, respectively. Received 5 July 2001/ Accepted in revised form 27 September 2001     

A latex agglutination test for the detection of Echinococcus multilocularis coproantigen in the definitive hosts

A latex agglutination test for detecting Echinococcus multilocularis coproantigen in definitive hosts was developed using latex beads sensitized with EmA9 monoclonal antibody raised against somatic antigens of adult E. multilocularis. A primary test (LA 1) was performed on 82 fecal samples of necropsied foxes, of which 46 were infected, and resulted in 61% sensitivity and 86% specificity. To increase the sensitivity, 4 ng/mL of excretory/secretory antigens of adult worms was added to the samples in a secondary test (LA 2), resulting in 91% sensitivity and 61% specificity. The positive predictive value of the LA 1 test and the negative predictive value of the LA 2 test were both 85%. The combination of the LA 1 and LA 2 tests is applicable and practical for use in situations that require quick diagnosis or screening based on the following interpretation: the samples that are positive in the LA 1 test are positive; the samples that are negative in the LA 2 test are negative; and the samples that are negative in the LA 1 test and positive in the LA 2 test are classified as suspicious.     

Identification and characterization of rhizobacteria isolated by enrichment culture on spinach roots

Rhizobacteria can be used for biological control and environmental restoration. In this study, we performed enrichment culture of rhizobacteria, identified isolates, and investigated the physiological properties of the bacterial isolates. Five bacteria differing in their colony morphology were isolated from spinach roots as enriched rhizobacteria. Four isolates were identified by sequencing of 16S rDNA as β and γ-Proteobacteria; 16S rDNA sequencing was not completed on one isolate. Based on microscopic observation, we determined that at least two types of bacteria differing in their morphology co-existed in this isolate, and that it may not be possible to culture the two types separately. Based on tests of substrate utilization, we could not find the characteristics that were common to the isolates. One of the five isolates was inoculated into non-sterile soil, and we examined its root-colonizing ability. The test strain which was not detected in the non-rhizosphere soil, accounted for about 20% of the total bacteria on the roots. These results suggested that enrichment culture might be useful for isolating bacteria with a high root-colonizing ability     

Similarities and differences in meat consumption patterns in Asia and Pacific-Rim countries

This study investigated consumer behavior towards meat across Asia and Pacific-Rim countries. Mail surveys were undertaken in 1994 and 1995. Data was obtained from 6197 households in Japan, the USA (mainland and Japanese–Americans in Hawaii), Australia, Thailand, Korea, Taiwan and China. The data were mainly analyzed using cluster analysis to determine the distance between each country and to classify countries into groups. Japanese respondents purchased beef more frequently than respondents from other countries. The average amount purchased on each occasion was far more for Australian respondents than for respondents from other countries, while Japanese purchased far less. Respondents in Japan, Korea, China and Australia thought beef was the most expensive meat, whereas lamb was thought to be the most expensive in the remaining countries. Chinese respondents indicated the highest consciousness of price indexes. The level of uneasiness about eating meat indicated by Asian respondents was relatively strong compared with respondents in Hawaii, the USA and Australia. Average ratings (semantic differential scale) on plans for future meat consumption were higher for beef than for other meats in Korea and China, whereas chicken was rated higher in the other countries. From the dendrogram, surveyed countries were classified into three groups: Japan, Taiwan and Korea; Thailand and China; and the USA, Japanese–Americans and Australia. Among them, the first and second clusters merged into a larger cluster, the 'Asian' group.     

Collaborative control initiatives targeting zoonotic agents of alveolar echinococcosis in the northern hemisphere

Alveolar echinococcosis is one of the most important lethal zoonotic helminth infections in the northern hemisphere. Currently, the threat to public health is increasing, as evidenced by the rising prevalence rate of alveolar echinococcosis, as well as the invasion of urban areas by infected wild foxes. This threat is further increased due to the involvement of pet dogs, and probably cats, as emerging sources of infection. These increased threats to public health also have associated economic risks; therefore, there is a need for effective and sustainable methods of control. In this paper, initiatives to control alveolar echinococcosis by targeting its definitive hosts through anthelmintic baiting campaigns initiated by local residents who used local resources for bait production, distribution and collection of fecal samples for diagnosis are described. Further, when such distribution programs are coupled with the use of GIS-based maps, the optimum distribution of bait was obtained. These programs have also included the use of intravital diagnostic analyses of infection rates, which have been overseen by the Forum on Environment and Animals (FEA), and also allowed a nationwide monitoring of echinococcosis in difinitive hosts. In addition, a government initiative requiring mandatory reporting of echinococcosis in dogs to health authorities was recently initiated in Japan. Overall, the results of this study have shown that use of collaborative control initiatives targeting zoonotic agents of alveolar echinococcosis can be an effective method for reducing the threat of lethal echinococcosis in the northern hemisphere.     

Characterization of royal jelly proteins in both Africanized and European honeybees (Apis mellifera) by two-dimensional gel electrophoresis

In this study, the proteins contained in royal jelly (RJ) produced by Africanized honeybees and European honeybees (Apis mellifera) haven been analyzed in detail and compared using two-dimensional gel electrophoresis, and the N-terminal amino acid sequence of each spot has been determined. Most spots were assigned to major royal jelly proteins (MRJPs). Remarkable differences were found in the heterogeneity of the MRJPs, in particular MRJP3, in terms of molecular weights and isoelectric points between the two species of RJ. Furthermore, during the determination of the N-terminal amino acid sequence of each spot, for the first time, MRJP4 protein has been identified, the existence of which had been only implied by cloning of its cDNA sequence. The presence of heterogeneous bands of glucose oxidase was also identified. Thus, the results suggest that two-dimensional gel electrophoresis provides a suitable method for the qualitative analysis of the proteins contained in RJ derived from different honeybee species.